Françoise Livolant

Precipitation of DNA by Polyamines: A Polyelectrolyte Behavior

Conditions of double-stranded DNA precipitation by the polyamines spermidine and spermine have been determined experimentally and compared to theoretical predictions. The influence of the concentrations of DNA and added monovalent salt, and of the DNA length has been investigated in a systematic manner. Three regimes of DNA concentrations are observed. We clarify the dependence of these regimes on the monovalent salt concentration and on the DNA length. Our observations make possible a rationalization of the experimental results reported in the literature. A comparison of the precipitation conditions of different kinds of polyelectrolytes suggests a general process. Our experimental data are compared to the ion-bridging model based on short-range electrostatic attractions. By starting from the spinodal equation, predicted by this model, and using the limiting form of Mannings fractions of condensed counterions, analytical expressions of the precipitation conditions have been found in the three regimes. Experimental and theoretical results are in good agreement.

DNA Aggregation Induced by Polyamines and Cobalthexamine

We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 μg/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.

DNA mesophases induced by spermidine: structural properties and biological implications.

Conditions of formation of DNA aggregates by the addition of spermidine were determined with 146 base pair DNA fragments as a function of spermidine and NaCl concentration. Two different phases of spermidine-DNA complexes are obtained: a cholesteric liquid crystalline phase with a large helical pitch, with interhelix distances ranging from 31.6 to 32.6 A, and a columnar hexagonal phase with a restricted fluidity in which DNA molecules are more closely packed (29.85 +/- 0.05 A). In both phases, the DNA molecule retains its B form. These phases are always observed in equilibrium with the dilute isotropic solution, and their phase diagram is defined for a DNA concentration of 1 mg/ml. DNA liquid crystalline phases induced by spermidine are compared with the DNA mesophases already described in concentrated solutions in the absence of spermidine. We propose that the liquid crystalline character of the spermidine DNA complexes is involved in the stimulation of the functional properties of the DNA reported in numerous experimental articles, and we discuss how the nature of the phase could regulate the degree of activity of the molecule.

Ordered phases of DNA in vivo and in vitro

In vitro, pure DNA forms multiple liquid crystalline phases when the polymer concentration is increased: precholesteric organization, cholesteric phase and columnar hexagonal phase. Similar organizations of chromatin can be found in vivo: hexagonal packing in bacteriophages and certain sperm heads, cholesteric organization in dinoflagellate chromosomes, bacterial nucleoids and mitochondrial DNA, helical-shaped chromosomes in many species. The different forms of condensed chromatin seem to be related to different local concentrations of DNA. In the highly condensed forms, chromatin is inactive and the double stranded DNA molecule is linear with small amounts of associated proteins.

Spermine-Induced Aggregation of DNA, Nucleosome, and Chromatin

We have analyzed the conditions of aggregation or precipitation of DNA in four different states: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), mononucleosome core particles (NCP), and H1-depleted chromatin fragments (ChF) in the presence of the multivalent cation spermine (4+). In an intermediate regime of DNA concentration, these conditions are identical for the four states. This result demonstrates that the mechanism involved is general from flexible chains to rigid rods and quasi-colloidal states. It is dominated by local electrostatic attractions that are considered, for instance, by the ion-bridging model. The onset of precipitation does not require the electroneutrality of the DNA chains. Above a given spermine concentration dsDNA aggregates remain neutral, whereas NCP aggregates turn positively charged. The difference is thought to originate from the extension of the positively charged proteic tails of the NCP. This suggests that local fluctuations of polyamine concentrations can induce either positively or negatively charged chromatin domains.

Salt-Induced Conformation and Interaction Changes of Nucleosome Core Particles

Small angle x-ray scattering was used to follow changes in the conformation and interactions of nucleosome core particles (NCP) as a function of the monovalent salt concentration C(s). The maximal extension (D(max)) of the NCP (145 +/- 3-bp DNA) increases from 137 +/- 5 A to 165 +/- 5 A when C(s) rises from 10 to 50 mM and remains constant with further increases of C(s) up to 200 mM. In view of the very weak increase of the R(g) value in the same C(s) range, we attribute this D(max) variation to tail extension, a proposal confirmed by simulations of the entire I(q) curves, considering an ideal solution of particles with tails either condensed or extended. This tail extension is observed at higher salt values when particles contain longer DNA fragments (165 +/- 10 bp). The maximal extension of the tails always coincides with the screening of repulsive interactions between particles. The second virial coefficient becomes smaller than the hard sphere virial coefficient and eventually becomes negative (net attractive interactions) for NCP(145). Addition of salt simultaneously screens Coulombic repulsive interactions between NCP and Coulombic attractive interactions between tails and DNA inside the NCP. We discuss how the coupling of these two phenomena may be of biological relevance.

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.

New observations on the twisted arrangement of Dinoflagellate chromosomes

The Dinoflagellate Prorocentrum micans has been studied in classical and high voltage transmission electron microscopy, with the help of a goniometric stage. The general structure of the nucleus is analysed with special reference to the links observed between chromosomes and the nuclear envelope, the nucleoplasm and the nucleolus. The chromosomes present stacked series of nested arcs which are studied in detail. The sense of the arcs can be changed by a simple tilt of the section. These arcs do not correspond to DNA filaments with a genuine bend but to an illusion created by the overlap of layers of filaments whose orientation turns along the chromosome axis. — The transversal orientation of DNA and the examination of defects allow to rule out the polytenic hypothesis. It is clear that this hypothesis does not apply to bacterial nucleoids, which however can form series of nested arcs as in Dinoflagellate chromosomes. — The twisted arrangement of Dinoflagellate chromosomes is that of a liquid crystal of the cholesteric type. DNA is known to self assemble into cholesteric phases and this affords informations on the origin of the elongated shape of chromosomes and on the mechanisms of condensation and aggregation observed in this particular chromatin.

Structure of toroidal DNA collapsed inside the phage capsid

The structure of DNA toroids made of individual DNA molecules of various lengths (3,000 to 55,000 bp) was studied, by using partially filled bacteriophage capsids in conjunction with cryoelectron microscopy. The tetravalent cation spermine was diffused through the capsid to condense the DNA under conditions that were chosen to produce a hexagonal packing. Our results demonstrate that the frustration arising between chirality and hexagonal packing leads to the formation of twist walls; the correlation between helices combined with their strong curvature impose variations of the DNA helical pitch.

A liquid crystalline phase in spermidine-condensed DNA

Over a large range of salt and spermidine concentrations, short DNA fragments precipitated by spermidine (a polyamine) sediment in a pellet from a dilute isotropic supernatant. We report here that the DNA-condensed phase consists of a cholesteric liquid crystal in equilibrium with a more concentrated phase. These results are discussed according to Florys theory for the ordering of rigid polymers. The liquid crystal described here corresponds to an ordering in the presence of attractive interactions, in contrast with classical liquid crystalline DNA. Polyamines are often used in vitro to study the functional properties of DNA. We suggest that the existence of a liquid crystalline state in spermidine-condensed DNA is relevant to these studies.